The present invention is directed to the MMSC1 gene, its protein product and the use of the protein to (i) detect mutant MMAC1 proteins, (ii) screen for drugs which can be used for suppressing tumor growth and (iii) identify proteins which interact with the MMAC1 gene or are involved in the tumor suppression pathway of the MMAC1 gene.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
A number of genetic alterations are involved in the oncogenesis of glioblastoma multiforme, including inactivation of p53, p16, RB, amplification of the gene encoding epidermal growth factor receptor and several other molecular alterations (Louis and Gusella, 1995). However the most common genetic alteration is the deletion of large regions or an entire copy of chromosome 10 (Fults et al., 1990; Rahseed et al., 1992). Recently, the tumor suppressor gene MMAC1 (Steck et al., 1997), also known as PTEN (Li et al., 1997) or TEP1 (Li and Sun, 1997) was mapped to 10q23 and shown to be mutated in 17-24% of xenografted and primary glioblastomas, 14% of breast cancer samples and 25% of kidney carcinomas (Steck et al., 1997). The mutation frequency in established cell lines of these tumor types is somewhat higher. In addition to this predicted involvement in sporadic cancer, germ-line MMAC1 mutations have been detected in two autosomal dominant disorders, Cowden disease (Nelen et al., 1997; Liaw et al., 1997), a syndrome that confers an elevated risk for tumors of breast, thyroid and skin, and Bannayan-Zonana syndrome (Marsh et al., 1997), a condition characterized by macrocephaly, lipomas, intestinal hamartomatous polyps, vascular malformations and some skin disorders. Mutations of MMAC1 in primary endometrial carcinomas (Kong et al., 1997) and in juvenile polyposis coli (Olschwang et al., 1998) have also been seen.
The predicted protein product of the MMAC1 gene has several regions of homology with other proteins. The MMAC1 protein has an animo terminal domain with extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions, and with auxilin, a protein involved in synaptic vesicle transport. The MMAC1 protein also has a region with extensive homology to protein tyrosine phosphatases (Steck et al., 1997; Li et al., 1997). Mutations of MMAC1 in tumors, its cytoplasmic localization (Li and Sun, 1997) and its intrinsic phosphatase activity (Li and Sun, 1997; Myers et al., 1997) suggested that its activity could be important in some aspect of tumor progression, possibly to counteract the oncogenic effect of a specific protein tyrosine kinase. In addition, MMAC1 is rapidly down-regulated by TGFxcex2 in cells sensitive to its cell growth and cell adhesion regulatory properties (Li and Sun, 1997).
Experiments on glioma cell growth have shown that MMAC1 is a protein phosphatase that exhibits functional and specific growth-suppressing activity. In such experiments, the introduction of HA-tagged MMAC1 into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing HA-tagged MMAC1 (Furnari et al., 1997). The ectopic expression of MMAC1 alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression (Furnari et al., 1997). Although these activities of MMAC1 are known, the mechanisms of tumor suppression by MMAC1 and the interaction of the MMAC1 protein with other proteins are not well understood.
Many cytosolic signaling proteins and cytoskeletal proteins are composed of modular units of small protein-protein interactive domains that allow reversible and regulated assembly into larger protein complexes. These domains include the Src-homology SH2 and SH3 domains (Schlessinger, 1994; Pawson, 1994), pleckstrin-homology (PH) domains (Lemmon et al., 1996; Shaw, 1996), phosphotyrosine-binding (PTB) domains (Harrison, 1996; van der Greer and Pawson, 1995; Kavanaugh et al., 1995) and postsynaptic density protein, disc-large, zo-1 (PDZ) domain (Woods and Bryant, 1991; Dho et al., 1992; Woods and Bryant, 1993; Kennedy, 1995; Kornau et al., 1995). So far, PDZ domains have been found in more than 50 proteins (Tsunoda et al., 1997), and many proteins have multiple PDZ domains (Pawson and Scott, 1997). For a review of PDZ domains, as well as the other protein-protein interactive domains, see Pawson and Scott (1997).
A distinguishing feature of PDZ domains is their recognition of short peptides at the carboxyl terminal end of proteins. For example, one family of PDZ domains selected peptides with the consensus motif Glu-(Ser/Thr)-Xaa-(Val/Ile) (SEQ ID NO:1) at the carboxy terminus, whereas a second family of PDZ domains selected peptides with hydrophobic or aromatic side chains at the carboxy terminal three residues (Songyang et al., 1997). The presence of multiple PDZ domains in proteins may have at least two important consequences. An individual PDZ-containinig protein could bind several subunits of a particular channel thereby inducing channel aggregations. Furthermore, the individual domains of a protein can have distinct binding specificities thereby inducing the formation of clusters that contain heterogeneous groups of proteins.
One example of this latter consequence of multiple PDZ domains is the InaD protein which contains five PDZ domains and acts as a scaffolding protein to organize the light-activated signaling events in Drosophila (Shieh and Zhui, 1996; Tsunoda et al., 1997). InaD associates through distinct PDZ domains with a calcium channel(TRP), phospholipase C-xcex2 (the target of rhodopsin-activated heterotrimeric guanine nucleotide-binding protein (Gqxcex1)) and protein kinase C.
Two further properties of PDZ domains or proteins which contain them may expand their potential activities. First, some PDZ domains may bind internal peptide sequences and, indeed, have a propensity to undergo homotypic or heterotypic interactions with other PDZ domains (Brenman et al., 1996). Second, proteins with PDZ domains frequently contain other interaction modules, including SH3 and LIM domains, and catalytic elements such a tyrosine phosphatase or nitric oxide synthase domains. PDZ domains may therefore both coordinate the localization and clustering of receptors and channels, and provide a bridge to the cytoskeleton or intracellular signaling pathways.
It is desired to determine the mechanisms of tumor suppression for MMAC1 and to identify proteins which interact with the MMAC1 protein. Such proteins can be used to assay for mutated MMAC1 proteins and/or screen potential drugs for suppressing tumor growth and/or identify additional proteins which interact with MMAC1.
The present invention is directed to the MMSC1 gene, its protein product and the use of the protein to (i) detect mutant MMAC1 proteins, (ii) screen for drugs which can be used for suppressing tumor growth and (iii) identify proteins which interact with the MMAC1 gene or are involved in the tumor suppression pathway of the MMAC1 gene.
Using yeast two-hybrid screening, it has been found MMAC1 binds to a protein herein named MMSC1. The nucleotide sequence is set forth as SEQ ID NO:2, and the amino acid sequence is set forth as SEQ ID NO:3. It has been found MMSC1 has 11 PDZ domains and that one or more of these domains interacts specifically with the three carboxyl terminal amino acids of MMAC1. Specifically, it has been found that PDZ domain number 7 interacts with MMAC1. Since MMSC1 contains 11 PDZ domains and interacts with MMAC1, known tumor suppressor having a region of homology with protein tyrosine phosphatases, MMSC1 acts as a scaffolding protein in a common biochemical pathway with MMAC1. These characteristics indicate that the interaction between MMAC1 and MMSC1 is required for the tumor suppressor activity of MMAC1.